The ryanodine receptor (RyR) is comprised of 4 RyR protomers and proteins that bind to the cytoplasmic domain of the channel forming a macromolecular signaling complex. This .proposal addresses the mechanisms by which allosteric modulators RyR function. Two specific forms of allosteric modulation will be examined: 1) regulation of the channel by derivatives of 1,4-benzothiazepines that potently effect channel gating via allosteric effects; 2) regulation of the channel by protein-protein interactions with the stabilizing subunit FKBP12/12.6 (calstabinl/2). The project will involve extensive collaborations with the PI of Project I (Arthur Karlin) and will use chemicals synthesized in Core B and animal models and cell culture in Core C. Four aims are proposed: Aim 1: Allosteric regulation of RyR2 by small molecules that enhance binding of calstabin2 to RyR2. Effects of 1,4-benzothiazepine derivatives on RyR2 channel function will be examined using RyR2 channels reconstituted into planar lipid bilayers. With Dr. Karlin (Project I) the binding site on RyR2 for the 1,4-benzothiazepine derivatives will be identified using phptoaffinity radiolabels synthesized by the chemistry Core B. The hypothesis is derivatives of 1,4-benzothiazepines bind to and allosterically modulate the function of RyR2 and RyRI. Aim 2: Allosteric modulation of RyR2 as a mechanism for preventing cardiac arrhythmias RyR2 are;PKA hyperphosphorylated and "leaky" in atrial fibrillation (AF) and JTV519 prevents exercised induced cardiac arrhythmias in WT and calstabin2+/- mice but not in calstabin2- /- mice indicating that the mechanism of action of this novel anti-arrhythmic drug requires calstabin2. The hypothesis is small molecules that enhance calstabin2 binding to RyR2 can prevent cardiac arrhythmias via allosteric modulation of RyR2. Using genetic mouse models harboring RyR2 mutations linked to sudden cardiac death in humans, and RyR2 mutations that mimic constitutively PKA phosphorylated or non- phosphprylatable RyR2 and animal models of AF, and myocardial infarction, we will determine whether enhancing binding of calstabin2 to RyR2 prevents cardiac arrhythmias. Aim 3: Stabilization of calstabin2 binding to RyR2 as a mechanism for treating heart failure (HF). RyR2 are PKA hyperphosphorylated and depleted of calstabin2 in HF and'JTV519 improves cardiac function in WT and calstabin2+/- mice but not in calstabin2-/- mice. Using genetic mouse models and models of myocardial infarction, we will determine whether enhancing binding of calstabin2' to RyR2 using JTV519 that modify RyR2 via allosteric effects improve cardiac function in HF. Skeletal muscle fatigue is increased and RyR1 are PKA hyperphosphorylated and depleted of calstabinl in HF, and JTV519 induces rebinding of calstabinl to RyRI probably via an allosteric effect on the channel. Using'animal models of myocardial infarction and HF, we will nvestigate whether JTV519 improves skeletal muscle function in HF. The studies are significant because they may lead to a novel therapeutic approach based on allosteric modulation of RyR that can result in mproved therapy for human cardiovascular diseases.